Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

Leishmania parasites include important pathogens and model organisms are even used for the production of recombinant proteins. However, functional genomics characterization essential genes often limited in because low-throughput technologies gene disruption or tagging absence components RNA interference. Here, we tested T7 polymerase-dependent CRISPR-Cas9 system by Beneke et al. glmS ribozyme-based knock-down parasite tarentolae. We successfully deleted two reference encoding flagellar motility factor Pf16 salvage-pathway enzyme adenine phosphoribosyltransferase, resulting immotile drug-resistant parasites, respectively. In contrast, were unable to disrupt mitochondrial flavoprotein Erv. Cultivation L. tarentolae standard BHI medium resulted a constitutive down-regulation an episomal mCherry-glmS reporter 40 60%. For inducible knock-downs, evaluated growth alternative media identified supplemented MEM, IMDM McCoy’s 5A as candidates. MEM allowed inducible, glucosamine concentration-dependent more than 70%. chromosomal glmS-tagging Pf16, phosphoribosyltransferase Erv did not reveal phenotype. Our data demonstrate suitability well limitations system, which was restricted moderate efficiencies knock-downs caused no detectable phenotype knock-downs.